Double-label immunofluorescence of arterial innervation among muscles in the lateral plantar compartment of the rat hind paw. Red labeling is with secondary antibodies conjugated with Cy3, green with Cy2. The images in A and B are from a larger source artery and those in C-H are from smaller tributaries. L = arterial lumen, TA = tunica adventitia, TM = tunica media, M = skeletal muscle. Scale bar = 25 μm. A, B. Cross section of a larger artery with lumen (L) toward the right. Anti-CGRP (A) labeled sensory innervation that is located primarily in the tunica adventitia (green arrows). This CGRP-positive sensory innervation is supplied by axons that have thin calibers (small green arrows) and slightly thicker-calibers (large green arrows). Double labeling with anti-NPY (B) revealed sympathetic innervation concentrated at the border between the tunica media and tunica adventitia (red arrows). C, D. Cross section of an arteriole with lumen to the upper right, Muscle fibers (M) in cross section below. Double staining with anti-ASIC3 (C) and anti-NF200 (D) demonstrated that ASIC3 is expressed primarily on the slightly thicker caliber innervation that is NF200-positive (yellow arrows). E, F. Small arterioles viewed both in cross section (lumen labelled L) and on edge (oval structure labelled TM). Double labeling with anti-ASIC3 (E) and anti-CGRP (F) revealed that ASIC3 is coexpressed exclusively with CGRP, primarily on the thicker-caliber innervation (yellow arrows). The smaller-caliber CGRP innervation is usually ASIC3-negative (green arrows). G, H. Edge of an artery cross section; lumen is out of the picture to the right; tunica media (TM) and tunica adventitia (TA) are marked. Double labeling with anti-TRPV1 (E) and anti-CGRP (F) demonstrated that TRPV1 is also coexpressed on the larger caliber CGRP-positive innervation (yellow arrows).
Molliver et al. Molecular Pain 2005 1:35 doi:10.1186/1744-8069-1-35